We report on the molecular characterisation of two novel granule proteins of the protozoon and human pathogen Entamoeba histolytica. The proteins, which were named grainin 1 and 2, show a considerable structural similarity to calciumbinding proteins, particularly within EF-hand motifs. Each grainin possesses three of these putative calcium-binding sites. Based on careful inspection of known structures of protein families containing EF-hands, a domain of grainin 1 covering two EF-hand motifs was modeled by homology. Calcium-binding activity of grainins was demonstrated by two independent methods. These granule proteins may be implicated in functions vital for the primitive phagocyte and destructive parasite such as control of endocytotic pathways and granule discharge. ß

The protozoan parasite Entamoeba histolytica contains a second antibacterial protein with lysozyme-like properties. The newly recognized bacteriolytic protein was purified from extracts of amoebic trophozoites to allow amino-terminal sequencing. Subsequent molecular cloning revealed that it is an isoform of the amoeba lysozyme described previously but also demonstrated a substantial sequence divergence of the two forms. As lysozymes typically are basic proteins, the novel amoebic protein differs markedly in having a pI of 4.5. There is no significant similarity of both amoeba lysozymes with any bacteriolytic protein of other organisms reported so far ; however, striking sequence identity is found with predicted gene products of unknown function derived from the bacteria-feeding nematode Caenorhabditis elegans.z 1998 Federation of European Biochemical Societies.

Amoebapore, a 77-residue peptide with pore-forming activity from the human pathogen Entamoeba histolytica, is implicated in the killing of phagocytosed bacteria and in the cytolytic reaction of the amoeba against host cells. Previously, we structurally and functionally characterized three amoebapore isoforms in E. histolytica but recognized only one homolog in the closely related but non-pathogenic species Entamoeba dispar. Here, we identified two novel amoebapore homologs from E. dispar by molecular cloning. Despite strong resemblance of the primary structures of the homologs, molecular modeling predicts a species-specific variance between the peptide structures. Parallel isolation from trophozoite extracts of the two species revealed a lower amount of pore-forming peptides in E. dispar and substantially higher activity of the major isoform from E. histolytica towards natural membranes than that from E. dispar. Differences in abundance and activity of the lytic polypeptides may have an impact on the pathogenicity of amoebae.

As previous reports suggested that a hyaluronidase is involved in tissue invasion of Entamoeba histolytica, we searched for such an activity in trophozoite extracts. A hyaluronidase activity was not detectable in long-term cultures or in amoebae freshly passaged through a gerbil liver, as evidenced by four different techniques.Entamoeba histolytica is the causative agent of human amoebiasis. Worldwide the protozoan parasite causes about 50 million cases of colitis or extraintestinal abscesses, and the disease results in at least 50,000 fatalities annually (33). The reproductive forms, the trophozoites, inhabit the cavity of the lower intestine in humans. For unknown reasons, they invade the mucosa of the colon and can enter several extraintestinal organs via the blood circulation system. Several factors are considered to play a crucial role in destroying the colonic epithelium: the adherence to cells is mediated by a Gal-and GalNac-specific lectin (24,26). Subsequently, cells are killed by pore-forming peptides termed amoebapores (19,20). Extracellular matrix proteins are degraded by the proteolytic activity of several isoforms of cysteine proteinases (3,14,27,30). Conceptually, for the effective spread of the parasite, amoebae should exert hyaluronidase-like activities. Therefore, the presence of such an enzyme in trophozoite extracts may be postulated.The substrate of a hyaluronidase is hyaluronic acid (hyaluronan), a nonsulfated glucosaminoglycan that consists of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine and which is a major structural component of the interstitial matrix of connective tissue in the lamina propria of the gastrointestinal tract (18). Hyaluronidases are broadly distributed enzymes and have been implicated in tissue invasion (5, 7, 17). Parasite-associated hyaluronidases are thought to participate in the infective process. In two parasitic nematodes, Ancylostoma caninum and Anisakis simplex, hyaluronidase activity is present in the gastrointestinal invasive stage and may facilitate tissue histolysis and mucosal invasion (11,12). Hyaluronidase activity in E. histolytica extract has been described previously (2, 16, 32). Activity was detected by measuring the loss of viscosity or turbidity of hyaluronic acid and the release of N-acetylglucosamine after incubation with crude trophozoite extract. However, the results are equivocal. It is reported that hyaluronidase activity is present only in amoebic isolates freshly passaged through a hamster liver. After a few weeks of cultivation, the activity is lost (2). Considering that hyaluronidase is expressed only during the invasion process and is rapidly lost when the appropriate stimulus is absent, the finding that hyaluronidase activity is present in stock cultures of E. histolytica without passaging through the livers of hamsters is controversial (16).Despite the fact that the existence of hyaluronidase activity in E. histolytica was reported decades ago and the possibility that such a factor plays a essential role i...

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